In our previous studies we had demonstrated that arsenic (As3+), an environmental metalloid metal, was able to induce transformation of the human bronchial epithelial cells. Additional experimental data revealed presence of cancer stem-like cells (CSCs) among the transformed cells induced by As3+. Other preliminary data showed that: (i) The CSCs induced by consecutive low-concentration As3+ treatment of the human bronchial epithelial cells exhibited significant decrease of reactive oxidative species (ROS) due to severe inhibition of the mitochondrial oxidative phosphorylation (OXPHOS); (ii) As3+ induced JNK and STAT3 (pSTAT3S727) phosphorylation in mitochondria along with a diminish of the mitochondrial transcription factor A (TFAM); (iii) integrated transcriptomic and metabolomic analyses demonstrated a higher rate of glycolysis and lower levels of mitochondrial metabolism due to mitochondrial DNA (mtDNA) depletion among these As3+-induced CSCs; and (iv) a unique glycolytic feature that is different from nave embryonic stem cells (ESCs) and cancer cells was found in these As3+-induced CSCs. Both ESCs and cancer cells direct glycolysis for lactate production. In contrast, the As3+-induced CSCs show increased conversion of the glycolytic intermediates into the subsidiary pathways for the generation of N-acetylglucosamine important for O-GlcNAcylation of the stemness genes and the S-adenosyl methionine (SAM) that contributes to DNA and histone methylation. Accordingly, the goal of this application is to determine: (1) is As3+-induced JNK-dependent pSTAT3S727 responsible for the inhibition of mitochondria; (2) if so, how this JNK-dependent pSTAT3S727 signaling pathway elicited by As3+ impairs the integrity or function of mitochondria, such as mtDNA replication, transcription, OXPHOS, etc; and (3) how the impaired function of mitochondria contributes to the generation of the CSCs induced by As3+. We hypothesize that As3+-induced JNK-dependent pSTAT3S727 signaling promotes formation of the CSCs by inhibiting mitochondrial OXPHOS and ROS generation, and the subsequent enhancement of glycolysis of the cells. To test this hypothesis, the following three specific aims are proposed: Specific Aim 1: determine how As3+ activates mitochondrial JNK that phosphorylates STAT3 S727 (pSTAT3S727) in BEAS-2B and other lung cells for the formation of CSCs. We will focus on the activation of mitochondrial-localized upstream kinases of JNK in response to As3+. The JNK dependent phosphorylation of additional mitochondrial proteins will be investigated through mitochondrial phosphoproteome; Specific aim 2: understand how As3+-induced JNK-dependent pSTAT3S727 inhibits mitochondria by addressing the role of pSTAT3S727 in mtDNA binding, its interaction with the mitochondrial transcription factor A (TFAM), and its effects on mitochondrial ROS production, proteome and the cellular metabolomics in the As3+-treated cells and As3+-induced CSCs; Specific Aim 3: utilize our unique mouse orthotopical lung cancer model, together with clinically relevant targeting agent, to preclinically explore therapeutic potential of inhibitors of JNK, STAT3 and glycolysis in CSC-generated lung cancer model in mice. Both short- and long-term systemic regimens of JNK, STAT3 and glycolysis inhibitors will be tested. We anticipate that the results from the proposed studies will unravel importance of As3+-induced JNK-dependent pSTAT3S727 on the generation of CSCs and lead to emerging of new concepts of As3+ carcinogenesis by emphasizing the ability of As3+ in CSC induction. Moreover, we believe that the date generated from completion of this project will be of real value in defining some new and straightforward targeting points that may help accelerate their use in clinical settings.